Evaluating in vitro antioxidant activity and GC-MS analysis of Scoparia dulcis Linn ( Scrophulariacae )

Article history: Received on: 09/04/2015 Revised on: 07/05/2015 Accepted on: 21/05/2015 Available online: 27/07/2015 Free radical scavenging activity was observed in both the methanol extract (M.E) and aqueous extract (A.E) of Scoparia dulcis respectively. In this study significant free radical scavenging activity was determined by evaluating the inhibition concentration (IC50) in each test. In 2, 2-diphenyl-1-picrylhydrazyl (DPPH) model the extract displayed potential free radicals scavenging activity with IC50 of M.E is 311.13μg/mL and A.E is 441.96μg/mL. Nitric oxide model displayed IC50 of 293.77μg/mL in M.E and 434.93μg/mL in A.E. While superoxide ion model showed IC50 of 281.02 μg/mL and 440.14μg/mL respectively for both methanol and aqueous extract when compared to standard ascorbic acid. The presence of phenol, flavonoid and total antioxidant in both the extract justifies the antioxidant potential of the plant which brings about its free radicals scavenging potential. GC-MS analysis showed the presence of 6 different phytochemicals with (Z)-7Hexadecenyl acetate found to be the compound with maximum peak percentage 51.51% in M.E and βCyclocitral with 43.90% in A.E respectively. Thus we conclude that the antioxidant activities may be due to the cumulative effect of the phytochemicals present in the plant which genuinely designate them as free radical scavenger.


INTRODUCTION
Free radicals play a dual role as they can be either harmful or helpful to the body (Pham-Huy et al., 2008).So, it will be appropriate to examine the possible role of free radicals in disease and most importantly, harnessing the therapeutic phytochemicals from Scoparia dulcis against these pro-oxidants.It is well documented that a number of physiological processes in human body lead to the generation of a series of oxygen-centered free radicals namely reactive oxygen species (ROS) and reactive nitrogen species (RNS) as by-products.However, imbalance in their production impairs the innate antioxidant defense system of the cell, resulting in peroxidation of unsaturated fatty acids, membrane protein damage (proteins, carbohydrates denaturation) and DNA mutation (nucleic acids denaturation) causing oxidative/nitrosative stress (Maes et al., 2011) which ultimately initiate the genesis of many multifactorial diseases.While the use of synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), Propylgallate (PG) and butylated hydroquinone have often been implicated to achieve immediate result, recent data indicates that these synthetic antioxidants could have carcinogenic effects thus fueling an intense search for newer and efficient antioxidants (Yevgenia et al., 2013).S. dulcis commonly called as sweet broom weed is a perennial herb, widely distributed in tropical and subtropical regions.Traditionally it has been used as remedies for varieties of ailments like stomach troubles, hypertension, diabetes, inflammation, bronchitis, hemorrhoids, hepatosis, analgesic, diuretic, antipyretic and cytotoxicity (Freire et al., 1993;Hayashi et al., 1993;Ahsan et al., 2003).Phytochemical screening revealed that S. dulcis contains a number of active principles such as diterpenoids, flavonoids, tannins, alkaloids, triterpenes, hexacosonol, β-sitosterol, ketonedulcitone, ammelin, glutinol and scoparinol (Satyanarayana, 1969;Edeoga et al., 2005;Chow et al., 1974;Freire et al., 1993;Ahmed et al., 2001).In addition, other active principles such as scoparic acid A&B, scopadulcic acid A&B, scopadulciol and scopadulin (Hayashi et al., 1990) have also been identified and all these isolated phytochemical have shown to contribute to the medicinal properties of the plant.Thus, showing the potential medicinal efficacy of each extracted compounds present in the plant, permits not only the demonstration of their physiological activity but also facilitates pharmacological studies leading to synthesis of a more potent drug with reduced toxicity (Manna and Abalaka, 2000).Hence, it is important to determine the antioxidant efficacy of S. dulcis and also for its phytochemicals constituents.

Collection and identification
The plant S. dulcis purchased from IMPCOPS, Chennai and was authenticated by Dr. D Aravind, National institute of Siddha, Department of Medicinal Botany.Voucher specimens have been deposited at the Herbarium of the institute, Reg no NIS/MB/62/2012.30 g of S. dulcis leaves were extracted with 250 mL of sterile distilled water and 70% methanol respectively using the Soxhlet apparatus at 60 • C. The extract was then filtered with Whatman No 1 filter paper and then freeze dried stored at 4˚C for further investigation.The extraction efficiency was quantified by determining the weight of each of the extracts and the percentage yield was calculated.

Total antioxidant capacities
Total phenolic and flavonoids of the contents of the extracts were determined by method described by Demiray et al., (2009) and Wang et al., (2000) respectively.Total antioxidant capacities of both extracts were evaluated as per the method described by Prieto et al., (1999)

Free radical scavenging activities
The free radical scavenging capacities of the extracts were determined using DPPH model according to Blois (1958).The nitric oxide and superoxide anion scavenging assays were carried out according to the method of Sreejayan et al., (1997) andLiu et al., (1997) Reducing power was carried out by the method of Yildirim et al., (2001).

Gas chromatography
Agilent 6890 gas chromatograph was equipped with a straight deactivated 2 mm direct injector liner and a 15m Alltech EC-5 column (250μ I.D., 0.25μ film thickness).A split injection was used for sample introduction and the split ratio was set to 10:1.The oven temperature program was programmed to start at 35 o C, hold for 2 minutes, then ramp at 20 o C per minute to 260 o C and hold for 5 minutes.The helium carrier gas was set to 2 ml/minute flow rate (constant flow mode).

Mass Spectrometry
JEOL GC mate II bench top double-focusing magnetic sector mass spectrometer operating in electron ionization (EI) mode with TSS-2000 software was used for all analyses.Lowresolution mass spectra were acquired at a resolving power of 1000 (20% height definition) and scanning from m/z 25 to m/z 700 at 0.3 seconds per scan with a 0.2 second inter-scan delay.High resolution mass spectra were acquired at a resolving power of 5000 (20% height definition) and scanning the magnet from m/z 65 to m/z 750 at 1 second per scan.

Mass spectrometry library search
Identification of components from purified compound was match with their recorded spectra and compared with the data bank mass spectra of NIST library V 11 provided by the instruments software.

Extraction yield, total phenolic, flavonoid contents and total antioxidant activity
The recent growth in the knowledge of free radicals and reactive oxygen species (ROS) in biology is producing a medical revolution which promises a new age of health and disease management.The most common ROS/RNS includes superoxide anion (O 2 -), hydrogen peroxide (H 2 O 2 ), peroxyl radicals (ROO − ) nitric oxide (NO) could be produced in most cells when subjected to stress.So, naturally there is a need for dynamic balance between the amount of free radicals and antioxidants generated to help regulate proper homeostasis at the cellular level.The interest in the physiological role of bioactive compounds present in plants has increased dramatically over the last decade, particularly in relation to human health.The pharmacological effects exerted by polyphenols on the human body are thought to be strongly related with their high antioxidant capacity (Hainal et al., 2011).In our study both methanol and aqueous extract of S. dulcis displayed a considerable antioxidant activity which is justified by the content of polyphenol like phenol and flavonoids (table-2).
As reported by Mahakunakorn et al., (2004) these phenolics and flavonoids compounds present in extracts are believed to intercept the free-radical chain of oxidation and donate hydrogen from the phenolic hydroxyl groups, thereby forming stable free radicals, which do not initiate or propagate further oxidation.

Free-radical scavenging activity
DPPH, superoxide and nitric oxide radical scavenging activity stays one of the most widely used method for screening the antioxidant activity of plant extract.The extract displayed a creditable DPPH scavenging (Fig- 1) nitric oxide radical scavenging (Fig- 2) and superoxide ion scavenging activity (Fig- 3) for their respective extract.However, in the entire three tests methanol extract displayed better scavenging activity than aqueous for this study.These finding suggest that the plant extract could have contain phytochemicals that is capable of donating hydrogen to a free radical in order to remove the odd electron which is responsible for the radical's reactivity.The IC 50 value for both the extract in DPPH, nitric oxide and superoxide ion scavenging model were tabulated in table-3.It is observed that S. dulcis extracted with methanol significantly has the enhanced ability to scavenge free radicals, and this could be attributed to the fact that methanol being a polar solvent proved better than the non-polar aqueous solvent for extraction.The inverse correlation between total phenol and flavonoid content and IC 50 values in methanol and aqueous were also justified, because with higher total phenolic and flavonoid content in the plant, the lower the amount of extract is required to reduce the DPPH, nitric oxide and superoxide ion scavenging activity.These results were also found to be concomitant with the total antioxidant activity observed in this study for both the extract (table-2).This is in agreement with Rice et al., (1997) who reported that phenolic compounds and flavonoids have been associated with anti-oxidative action in biological systems, acting as scavengers of singlet oxygen and free radicals.To further supports our finding recent studies carried out by Pratap et al., (2014) and Arasan and Abdul, (2012) reported that various extracts of S. dulcis have showed potent free radical scavengers.Although the scavenging abilities of the extracts were lower than those of ascorbic acid standard, it was evident that the both methanol and aqueous extracts did show some proton-donating ability and this could serve as free radical inhibitors or scavengers, acting possibly as primary antioxidants.
Reducing properties of the antioxidants are generally associated with the quality of antioxidants activity of the plant.In this study a steadily increased in reducing capacity with direct proportion to the increasing concentration of the extract (fig- 4) where methanol, aqueous and ascorbic extract at 250μg/mL was found to be 0.426, 0.324 and 0.589 μg/mL (fig- 4).Reducing properties of the antioxidant are general associated with the presences of reductones and they exert anti-oxidant action by breaking the free radical chain and donating hydrogen atoms, thus preventing peroxides formation.As already discuss earlier, methanol being a potent extraction solvent could have extracted enhanced reductones content which again supports its potent reducing capacity when compared to that of aqueous solvent.
Our studies presented a positive relationship between the free radical scavenging activity the effective antioxidant capacity displayed by total phenol, flavonoids and antioxidant content in both methanol and aqueous extract which clearly justifies the therapeutic efficacy of S. dulcis extracts as an alternative to synthetic antioxidant.

GC-MS analysis of bioactive constituents
Knowledge of the chemical constituents of plants is desirable not only for the discovery of new therapeutic agents, but also disclosing new sources of economic phytocompounds for the synthesis of complex chemical substances and determining the actual significance of folkloric remedies (Milne et al.,1993).GC-MS analysis was carried out for both the extracts and results pertaining to these compounds (fig: 5 & 6) were identified through mass spectrometry attached with GC.Molecular weight, molecular formula and structure of the isolated compounds were ascertained.GC-MS analysis shows the presence of different phytocompounds in methanol extract (Table-4) namely 2-hexyldecanoic acid methyl ester (6.10%), dextromoramide (31.88%), (Z)-7-Hexadecenyl acetate (51.51%),Cis-5-eicosenoic acid methyl ester (3.98%), 2heptadecyl imidazoline (3.85%) and methyl eicosanoate (2.64%).The two most dominant compounds identified as dextromoramide with 31.88% belonging to opioid family are known to have analgesic activity this is in agreement with Ratnasooriya et al., (2003) who reported significant analgesic and the antihyperalgesic activity for S. dulcis decoction.The other most dominant compound (Z)-7-hexadecenyl acetate with 51.51% with no anti-oxidant activity reported.
Result tabulated in  for aqueous extract also shows the presence of different phytocompounds namely 1-Methyl-2-pyrrolidone-(4.83%), N1-Acetylspermine-(15.59%),L-(+)-ascorbic acid 2,6-dihexadecanoate-(22.71%), 3-Procaine- (6.74%), Cyclohexylamine-(6.20%) and beta-cyclocitral-(43.90%).The concentrated aqueous extract contains a variety of phytochemicals, among which the most dominant component of this plant with 22.71% is L-(+)-Ascorbic acid 2,6-dihexadecanoate and it has been reported to have antioxidant, anti-inflammatory and anti-nociceptive properties (Akinmoladun et al., 2007;Okwu and Emenike, 2006).Acetylspermine belonging to polyamines family, play essential role in the proliferation and development of mammalian cells.In addition, polyamines have been shown to exert antioxidant activity (Ha et al., 1998;Fujisawa, 2005).Phytochemical investigation of S. dulcis carried out so far, has reported terpenes such as triterpenes (Tsai et al., 2011), benzoylated diterpenes (Ahsan et al, 2003) to be the most commonly isolated compound but never a monoterpenes has been reported isolated from this plant.The terpene identified in this .particular study is a monoterpenes β-cyclocitral with the highest peak percentage of 43.90%.As such there has been no antioxidant activity recorded by β-cyclocitral however, it has been reported to have dissolution effect on cyanobacterial cell walls and membranes (Ozaki et al., 2009).Recent publication on S. dulcis aqueous extract via HPTLC analysis established a class of compounds including terpenoids, flavonoids, stilbenes, phenolic compounds and proanthocyanidins (Wankhar et al., 2015) CONCLUSION Thus summarizing these results, "it is evident that methanol extract of S. dulcis proved to have superior antioxidant capacity when compared to aqueous extract in this particular study and this may have resulted due to the greater extraction capacity of methanol when used as solvent.Hence, the possibility of using a .crude extract as an antioxidant would greatly reduce the need to obtain pure compounds via expensive industrial purification techniques.Further in depth toxicity and dosage may reveal its efficacy of the plant as an alternative to anti-oxidant therapy.

ACKNOWLEDGEMENT
The financial assistance provided by the UGC (UPE-Phase II) from University of Madras and University Grants Commission-RGNF is gratefully acknowledged.The author is grateful to University of Madras for providing the infrastructure to conduct the research.

Fig. 1 :
Fig. 1: Comparative DPPH scavenging activities of Scoparia dulcis extract and ascorbic acid.Values are the mean of duplicate experiments and represented as mean ± SD.

Fig. 2 :
Fig. 2: Nitric oxide scavenging activities of Scoparia dulcis extract and ascorbic acid.Values are the mean of duplicate experiments and represented as mean ± SD.

Fig. 3 :
Fig. 3: Superoxide anion scavenging activities of Scoparia dulcis extract and ascorbic acid.Values are the mean of duplicate experiments and represented as mean ± SD.

Fig. 4 :
Fig. 4: Comparative reducing power capacity of Scoparia dulcis extract and ascorbic acid.Reducing power compared between methanol, aqueous extract of S. dulcis and ascorbic acid respectively.

Table 1 :
Yield of methanol and aqueous extract of Scoparia dulcis.

Table 2 :
Total antioxidant, phenol and flavonoid content of S. dulcis plant extracts.Values are the mean of duplicate experiments and represented as mean ± SD.

Table 3 :
IC50 values of different extracts of S. dulcis in DPPH, Nitric oxide (NO) and superoxide ion scavenging assay.

Table . 4
: Bioactive components identified in Methanol extract of S. dulcis.

Table 5 :
Bioactive components identified in aqueous extract of S. dulcis.